ABSTRACT Metastasis accounts for more than 90% of all cancer deaths. The underlying mechanisms controlling the metastatic cascades are still poorly understood. Mitochondrial metabolism has emerged as a key component and potential master regulator of cancer metastasis, and increased mitochondrial activities have been observed in circulating tumor cells. We have previously developed a micropatterned tumor-stromal assay (TSA) to recapitulate spatially-resolved cellular cross-talks at the tumor-stroma interface in breast cancer, and we recently discovered that heterogeneous mitochondrial phenotypes are induced near the tumor-stroma interface. Our central hypothesis is that physicochemical interactions in tumor microenvironment give rise to heterogeneous mitochondrial and metabolic activities, leading to differential invasive and metastatic phenotypes. Here we will develop TSA-based tumor models to mimic critical tumor microenvironmental cues, and to evaluate their roles in regulating mitochondrial heterogeneity and metastatic potential both in vitro and in vivo. The proposed study will provide not only a systems understanding of the microenvironmental determinants of mitochondrial heterogeneity, but also the mechanistic relationship between mitochondrial and metastatic phenotypes. The insights and developed platform will advance targeted therapeutics for cancer metastasis, and benefit a broad range of areas including cancer metabolism, stem cell biology, biomarker discovery, and drug screening.